To try to develop highly sensitive, non-invasive diagnostic methods, we have been evaluating polymerase chain reaction (PCR) using primers based on the major surface glycoprotein (MSG) genes of human Pneumocystis. This is a family of genes that are closely related and that encode an important surface protein of Pneumocystis. PCR using primers based on this gene is potentially a highly sensitive method since this is a multicopy gene (estimated at approximately equal to 50-100 copies/genome). We have been evaluating the diagnostic potential using a conserved region of the gene family. Our studies have shown that the sensitivity of MSG-based primers is greater than that of previously utilized primers. We are currently evaluating these primers prospectively in collaboration with the Microbiology Department and investigators at SFGH using oral washes as the clinical specimen. Because human Pneumocystis cannot be cultured, we cannot directly determine if resistance to commonly used therapeutic agents is developing. However, molecular techniques can be used to identify mutations that may confer resistance in genes that are targets of therapeutic agents. The most commonly used agent to treat Pneumocystis pneumonia is the combination of trimethoprim, which targets dihydrofolate reductase (DHFR), and sulfamethoxazole, which targets dihydropteroate synthase (DHPS). We have cloned the human Pneumocystis DHFR gene, and have examined (by PCR and sequencing) the Pneumocystis DHFR and DHPS genes of a variety of human isolates from patients with Pneumocystis pneumonia. DHPS mutations were found in about one-third of patients, while no mutations have been found to date in the DHFR gene. We have also expressed recombinant human Pneumocystis DHFR, characterized the kinetics of this enzyme, and developed a rapid screening assay for agents which target DHFR, by expressing the enzyme in a yeast system. We have also developed a rapid method for detection of DHPS mutations using SSCP (single strand conformational polymorphisms), and have examined a large number of samples for DHPS mutations, including organisms obtained from an Italian cohort. We have also developed and evaluated a new typing technique using tandem repeats that occur in an intron of the MSG gene. Further studies of a region of the MSG expression site are ongoing; we have been able to demonstrate that recombination occurs in human Pneumocystis. These studies should provide improved diagnostic methods for PCP, help to better understand the epidemiology of Pneumocystis infection, and insights into the reasons for therapy or prophylaxis failures.